Today, lung cancer is the primary cause of death from cancer in almost all developed countries, including Japan, although a variety of therapeutic methods have been improved. Further, in spite of increased opportunities for early detection of lung cancer, approximately 60,000 lung cancer patients die in Japan every year. The main cause of death of lung cancer patients treated with surgical therapy is distant metastasis. Distant metastasis, including intrapulmonary metastasis observed in non-small-cell lung cancer patients, is often observed among patients treated by surgical resection alone. This is because, in such patients' cases, micrometastatic foci are already present at the time of surgical resection. The prognosis of a non-small-cell lung cancer patient can be improved in some cases involving the use of postoperative adjuvant chemotherapy, which is performed to suppress micrometastasis recurrence. However, the effects of postoperative adjuvant chemotherapy for non-small-cell lung cancer patients treated with a radical surgery have been often controversial since the 1980s. Even in 2003, the effects of postoperative adjuvant chemotherapy were still not verified in terms of efficacy (Scagliotti G V, Frossati R, Torri V, Crino L, Giaccone G, Silvano G, Martelli M, Clerici M, Cognetti F, and Tonato M., J Nat Cancer Inst. (2003) 95: 1453-1461). In 2004, some reports on the effects of postoperative adjuvant chemotherapy were published and data showing the efficacy of the therapy were disclosed (Winton T, Livingston R, Johnson D, Rigas J, Johnston M, and Butts C, Cormier Y (2005) N Engl J Med 352: 2589-2597; Strauss G M, Herndon J, Maddaus M A, Johnstone D W, Johnson E A, Watson D M, Sugarbaker D J, Schilsky R L, and Green M R. (2004) Proc Am Soc Clin Oncol 23: 621; Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon J P, and Vansteenkiste J. (2004) N Engl J Med 350: 351-360).
Also, in 2004, it was reported that primary lung adenocarcinoma patients in stage I were confirmed to have improved prognoses as a result of postoperative adjuvant chemotherapy with oral tegafur-uracil (a mixed formulation with a molar ratio of 4:1; hereinafter referred to as “UFT”) compared with patients treated by surgery alone (Kato H, Ichinose Y, Ohta M, Hata E, Tsubota N, Tada H, Watanabe Y, Wada H, Tsuboi M, Hamajima N, and Ohta M. (2004) N Engl J Med 350: 1713-1721). However, only 15% of primary lung adenocarcinoma patients in stage IB can be expected to have improved prognoses as a result of postoperative adjuvant chemotherapy with oral tegafur-uracil. Therefore, if biomarkers that allow reasonable and objective prediction/determination of the prognosis of a patient treated with postoperative chemotherapy can be discovered in view of molecular biology, such biomarkers would be significantly useful.
Hitherto, as a method for identifying markers associated with drug efficacy, an immunobiochemical identification method has been known. In recent years, as a part of the post-genome study, an approach called pathological proteomics has been gaining attention. Specifically, it is a method comprising simultaneously analyzing actually expressed proteins in a blanket manner and searching for marker proteins for diseases. Examples of such pathological proteomics approach include a method using matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and a method using liquid chromatography ion trap mass mass spectrometer.
However, sufficient analysis performance cannot be realized by the above conventional pathological proteomics approach in the analysis of clinical pathology, by comparing protein expression patterns of biological samples from healthy individuals and those from patients, especially in a case where large variances in many factors are expected, and furthermore, a level of difference of each factor is too slight to distinguish it from an individual difference or a measurement error.
Therefore, in Patent Document 1, the present inventors reported a sample analysis method and a sample analysis program with excellent analysis performance, which allow pathological proteomics analysis. In addition, they reported “a lung adenocarcinoma lymph node metastasis diagnosis method and a diagnosis kit,” which have been completed with the use of the above method and program (Patent Document 2).
Non-muscle-type myosin IIA is a main constituent of the actomyosin cytoskeleton and known to be generally involved in the posterior-side contraction of cells during cell migration. Cellular projections were thought to be controlled via actin filament polymerization in a myosin IIA-independent manner. In recent years, it has been reported that myosin IIA is involved in formation of cellular projections associated with cell migration of non-cancer cells. However, the way in which myosin IIA functions during cell migration is unknown. Obviously, myosin IIA is involved in cell migration. However, in the past studies, protein profiles at expression levels observed during cell migration of cancer cells and isotypes of plural forms of myosin IIA were merely evaluated.
In recent years, clinical studies of non-small-cell lung cancer have showed that there is a correlation between the expression levels of myosin light chain kinase that causes myosin II activation and metastasis/recurrence, and that myosin IIA activation is a metastatic factor (Non-Patent Document 1). The important role of myosin IIA in metastatic cancer cells was suggested by an indirect study focusing on the small calcium-binding protein metastin 1. In many metastatic culture cell lines, the expression of metastin 1 is intensified and the metastatic nature is enhanced as a result of introduction of metastin 1 into culture cells (Non-Patent Document 2). Further, the main target molecule of metastin 1 is thought to be myosin IIA (Non-Patent Document 3). In addition, it has been shown that metastin 1 and myosin IIA coexist at an identical location in a tip portion of a migrating cancer cell (Non-Patent Document 4), and therefore metastin 1 influences phosphorylation caused by protein kinase, which is thought to control polymerization of myosin IIA and filaments (Non-Patent Documents 5 and 6).
Vimentin, which has a molecular weight of 57 kDa, is an intermediate filament protein that is expressed in the early phase of cell differentiation so as to be widely distributed. For all initial cell types, the expression of vimentin is observed. However, in many non-mesenchymal cells, vimentin is replaced by a different intermediate filament protein over the course of differentiation. Vimentin is expressed in a variety of mesenchymal cells such as fibroblasts and endothelial cells. There are several cell types of vimentin, which are derived from mesoderm and granulosa cells in mesothelia and ovaries. During the course of differentiation, epithelial cells acquire mesenchymal phenotypes that are essential for organogenesis in a reversible or irreversible manner. Epithelial-interstitial transition (interaction) in morphogenesis causes abnormal expression in a variety of epithelial cancers, including thyroid gland cancer, liver cancer, kidney cancer, prostate cancer, mammary gland cancer, and lung cancer, during tumorigenesis (Non-Patent Documents 7 and 8). Regarding general characteristics observed over the course of tumorigenesis, acquisition of interstitial phenotypes associated with loss of normal epithelial phenotypes, loss of polarizing epithelial morphology, and gradual acquisition of phenotypes (i.e., partial or entire motility and invasiveness) can be mentioned (Non-Patent Document 9).
Further, the collapse of epithelial morphology involves abnormal control of adhesion molecules, abnormal expression of N-cadherin, and epithelial-interstitial interaction (transition) with the expression of molecules such as fibronectin and vimentin in epithelial cells, such molecules originally being expressed in the interstitium. There is a correlation between abnormal expression of vimentin in tumors or transformants and increased motility, invasiveness, and poor prognosis (Non-Patent Documents 10 and 11). In lungs, vimentin is present in fibroblasts, smooth muscles, endothelia, and lymphoid lineage cells; however, it is not expressed in normal bronchial epithelial cells. A study conducted in recent years reported that vimentin-positive tumor cells in a fibrotic portion are mainly involved in tumor fibrosis accompanied by epithelial-interstitial transition (interaction), corresponding to the other study results (Non-Patent Document 12). In fact, acquisition of mesenchymal phenotypes relates to the expression of a protein serving as a mesenchymal marker and abnormal extracellular matrix deposition. Similar findings have been reported with the use of other models of, for example, progressive renal fibrosis in which the existence of vimentin-positive epithelial cells is associated with the degree of increase in fibrotic tissue (Non-Patent Documents 13 and 14).
Regarding the role of a marker, myosin has been known as a marker for differential diagnosis of rhabdomyosarcoma, and vimentin has been known as a marker protein for a variety of benign and malignant tumors such as malignant melanoma (Patent Document 3). Particularly recently, they have been studied as immunohistochemical markers for distinguishing epithelial malignant mesothelioma and lung adenocarcinoma (Non-Patent Documents 15 and 16).    Patent Document 1: WO 2004/090526    Patent Document 2: JP Patent No. 2006-53113    Patent Document 3: JP Patent No. 2006-518982    Non-Patent Document 1: Tumour Biol 26 153-157, 2005    Non-Patent Document 2: Oncogene 8 999-1008, 1993    Non-Patent Document 3: J Biol Chem 281 677-680, 2006    Non-Patent Document 4: J Biol Chem 278 30063-30073, 2003    Non-Patent Document 5: Biochemistry 44 6867-6876, 2005    Non-Patent Document 6: Biochemistry 42 14258-14266, 2003    Non-Patent Document 7: Nat Rev Cancer 2 442-454, 2002    Non-Patent Document 8: Cell 105 425-431, 2001    Non-Patent Document 9: J Cell Biol 135 1643-1654, 1996    Non-Patent Document 10: J Pathol 180 175-180, 1996    Non-Patent Document 11: Am J Pathol 150 483-495, 1997    Non-Patent Document 12: Lab Invest 84 999-1012, 2004    Non-Patent Document 13: Virchows 433 359-367, 1998    Non-Patent Document 14: Kidney Int 58 587-597, 2000    Non-Patent Document 15: The American Journal of Surgical Pathology 27(8) 1031-1051, 2003    Non-Patent Document 16: Pathology International 57 190-199, 2007